The toxic effects of physostigmine, an anticholinesterase drug, and its metabolite eseroline were investigated in three neuronal cell culture systems; mouse neuroblastoma N1E-115, rat _glioma C6 and neuroblastoma- glioma hybrid NG 108-15. Leakage of lactate dehydrogenase and release of [14C] adenine nucleotides were used as parameters of cell death. Physostigmine and eseroline (0.5 mM) elicited a time-dependent leakage of lactic acid dehydrogenase (LDH) from all three cell types. An increased release of [14C]adenine nucleotides was also detected from cells when they were prelabelled with [14C]adenine. Eseroline was comparatively more toxic than the parent compound, physostigmine. Eseroline elicited a dose- and time dependent leakage of LDH and release of adenine nucleotides from the neuronal cells. A nonneuronal cell line, rat liver ARL-15, was compar- atively the most resistant cell type; to eseroline toxicity. The con- centrations of eseroline needed for 50% release of adenine nucleotides or 50% leakage of LDH from NG-108-15 and N1E-115 cells in 24 hr ranged from 40 to 75 (mu)M. The concentrations of eseroline needed to obtain similar responses in C6 and ARL-15 cells were much higher and ranged from 80 - 120 (mu)M. Phase contrast microscopy showed extensive damage to three neuronal cell lines at concentration of eseroline as low as 75 (mu)M. The loss of ATP from N1E-115 cells exceeded 50% when they were treated with 300 (mu)M eseroline for 1 hr - at which time the leakage of LDH was not detectable. It seems that eseroline causes neuronal cell death by a mechanism involving loss of cell ATP. The formation of eseroline may contribute to the toxic effect of physostigmine.